Freeze etching and immunofluorescence were used to study the distribution and molecular content of tight and adherens junctions in the organ of Corti at the level of the reticular lamina. These studies indicate that tight junctions are found between all cell types in the reticular lamina, but the adherens junction is found only between supporting cells. Tight junctions (TJ) between hair cells and supporting cells have an exceptionally dense cytoplasmic undercoat and tortuous strand structure which may account for the TJ's ability to maintain its integrity during sound transduction in the absence of the adherens junction. Confocal fluorescence microscopy was used to determine the distribution of tight and adherens junction proteins. ZO- 1, a TJ protein, was present between all cell types in the reticular lamina. Adherens junction proteins, E-cadherin, alpha- and gamma- catenin, and p120, were present only in junctions between supporting cells. The adherens junction protein, beta-catenin, was found not only between supporting cells, but also between outer hair cells (OHCs) and supporting cells. Both the extensive cytoplasmic undercoat and the presence of beta-catenin in the TJ between OHCs and supporting cells suggest that the OHC tight junctions are enhanced, hybrid junctions. These junctions have a dual function as both permeability barriers and adhesion complexes and may derive at least part of their adhesive capacity from molecular components of the adherens junction. We also studied regulation of TJ permeability using the in vitro model system of MDCK cell monolayers and observed that the PKC activator, TPA, in the presence of Ca++ causes a decrease in transepithelial resistance. Freeze fracture analysis of these cells shows fragmentation and aggregation of tight junction strands. TPA treated cells show ZO1 staining in patches at the cell periphery while control cells show ZO1 completely lining the cell membrane. While we can associate ZO1 with the disruption of TJ structure it is not clear whether it is required for the reorganization of TJ structure and function.